JPH0669399B2 - Method for fractionating carboxyl-terminal peptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for conveniently separating a carboxyl-terminal peptide from a polypeptide.

[Problems to be Solved by Prior Art and Invention] In 1950, Edman developed a sequential amino acid residue sequencing method from the amino terminus of a peptide (P. Edman, Acta
Since Chem.Scand., 4 , 283 (1950)), various innovations have been made to date, and now, N-terminal sequence analysis can be performed using an automated sequencer with a very small amount of sample of picomole order. ("Seikagaku Chemistry Laboratory Lecture 2 Protein Chemistry", P.247-P.373, edited by the Japanese Biochemical Society, 1987, Tokyo Kagaku Dojin).

On the other hand, with the advent of genetic engineering technology in recent years, by determining the base sequence of DNA complementary to messenger RNA,
It has become common practice to predict the entire amino acid sequence of a polypeptide. However, even in this case, a part of the amino acid sequence of the desired polypeptide is known, and an oligonucleotide prepared based on this is used as a probe for complementary DN.
It is common to search for A, and if a probe corresponding to the amino acid sequence near the carboxyl terminus of the polypeptide can be used, the search will be easier. In addition, the polypeptide that actually exists may be processed, and in some cases does not contain all of the amino acid sequence encoded by the messenger RNA, the amino terminal of the polypeptide may be Determining the carboxyl terminus is extremely important.

At present, hydrazine decomposition method, tritium labeling method, and carboxypeptidase method are used as the method for analyzing the carboxyl terminus (“Secondary Chemistry Laboratory Lecture 2 Protein Chemistry”, P.230, Japan Biochemical Society, 1987. , Tokyo Kagaku Doujin).

Of these, the hydrazine decomposition method and the tritium labeling method are methods for determining only one amino acid residue at the terminal end of the carboxyl and cannot be used as a source of information for probe preparation.
It only gives low accuracy in identifying the position of the amino acid residue encoded by the messenger RNA.
The carboxypeptidase method utilizes various carboxypeptidases that sequentially cleave peptide bonds from the carboxyl terminus of a polypeptide,
Since the sequence is determined by tracking the change over time of the cleavage, (1) the operation is complicated, and (2)
The required sample amount is relatively large, and there are drawbacks such as (3) uncertainties in sequencing, and (4) not suitable for relatively long-chain polypeptides.

[Means for solving problems]

The present inventors have eagerly sought a carboxyl-terminal amino acid sequence analysis method that overcomes the drawbacks of these carboxyl-terminal amino acid analysis methods and provides a lot of reliable information from an extremely small amount of sample that is comparable to the amino-terminal sequence analysis method. The present invention has been achieved in the process of performing.

That is, the present invention specifically treats the polypeptide by cleaving the peptide bond between the lysine residue in the polypeptide and the subsequent amine acid residue on the carboxyl terminal side,
The resulting peptide mixture is reacted with a solid having a functional group capable of reacting with a free amino group on its surface to form a covalent bond, then between the amino-terminal residue of each peptide and its adjacent residue. The present invention relates to a method for fractionating a carboxyl-terminal peptide, which comprises collecting the released peptide by cleaving the peptide bond by acid treatment.

Hereinafter, the present invention will be described in more detail. According to the present invention, the carboxyl-terminal peptide binds to a solid as described below only at the amino-terminal α-amino group, whereas other peptides include amino-terminal amine groups as well as ε of lysine residues.
-Amino group also binds to a solid, and when this peptide-solid conjugate is treated with a suitable acid under suitable conditions, only the peptide bond between the amino-terminal residue and the adjacent residue is cleaved. Therefore, the carboxyl-terminal peptide utilizes the fact that the residue at the amino-terminal end remains in the solid and is released into the reaction solution, while the other peptides remain bound to the solid.

The most useful method for specifically cleaving the peptide bond between the lysine residue in the polypeptide and the subsequent amino acid residue on the carboxyl terminal side is Achromobacter lytic protease I [ Lysyl endopeptidase (EC3.4.21.50), hereinafter abbreviated as API]. This enzyme is extremely specific for the cleavage of Lys-X bond (X represents an amino acid residue), and there are very few non-specific cleavages ("Seikagaku Chemistry Laboratory 2 Protein Chemistry"). , Pp. 262, Japanese Biochemical Society, 1987, Tokyo Kagaku Dojin), and is particularly suitable for application to the present invention. Since API is a highly stable enzyme,
There are few restrictions on the conditions of the cleavage reaction, and the pH is 6 to 11, preferably 8 to 10.5, and the temperature is 4 to 50 ° C, preferably 20 to 45 ° C, and the molar ratio to the polypeptide to be cleaved is 1/20 to 1 /.
2000, preferably 1/200 to 1/600, is added and allowed to act for 1 to 50 hours, preferably 4 to 8 hours. As the buffer, various buffers commonly used in biochemical experiments can be used, but considering the influence on subsequent operations, a buffer having no free amino group is preferable, especially when Edman degradation is performed. The frequently used N-ethylmorpholine is preferred. Further, in order to smoothly proceed the cleavage reaction,
It is also preferred to loosen the higher order structure of the polypeptide without reducing the activity of the API, for which purpose eg 4-5 molar urea may be added.

In addition to API, endoproteinase Lys-C (trade name, Boehringer Mannheim) produced by Resobacter enzymogenes can also be used.

In addition, trypsin is an enzyme specific to lysine and arginine residues, and therefore, arginine residues in a polypeptide can be preliminarily identified, for example, in "Sequencing of proteins and p
eptides "G. Allen, P. 57-58, 1981, North-Holland Publis
The present invention can also be carried out by modifying the compound with cyclohexane-1,2-dione or the like by the method described in hing Company, Amsterdam; New York / Oxford, and then allowing tribucin to act in the same manner as API.

Functional groups capable of reacting with a free amine group to form a covalent bond include imide group, isourea, aldehyde group, cyano group, acetyl group, succinyl group, maleyl group, acetoacetyl group, dinitrophenyl group, trinitrobenzene. A large number of sulfonic acid groups, isothiocyanate groups, etc. can be mentioned, but the bond with the ε-amino group is stable even under the acid treatment conditions in which only the amino group is reacted and Edman decomposition proceeds. Isothiocyanato groups are preferred for the present invention. The preparation of a solid having a functional group such as an isothiocyanate group can be performed by, for example, “Sequencing of proteins and pep
tides "G. Allen, p.208,1981, North-Holl and Publishing
It can be carried out according to the method described in Company, Amsterdam; New York / Oxford. Examples of the solid carrier include porous glass, silica gel, polystyrene and the like.
Porous glass with a uniform pore size makes it easy to control the reaction,
Also, it is particularly preferable because it is hydrophilic, but in the case of polystyrene, which is a hydrophobic carrier, the hydrophilicity is increased by introducing a glucosaminol group into the isothiocyanate group (Iwanaga et al., Protein / Nucleic Acid).・ Enzyme 15 (10) 1052 (19
70)) It can be made easier to use. The coupling reaction between the solid having a functional group and the peptide mixture is pH 7-12, preferably 9-11, more preferably 9.5-10.5, and temperature 4-80.
The reaction is carried out at a temperature of preferably 10 to 60 ° C. for 5 minutes to 3 hours. At this time, it is preferable to replace the liquid with nitrogen to remove oxygen. After completion of the coupling reaction, a suitable volatile solvent,
For example, it is washed with a solvent such as acetonitrile or propanol, dried, and then treated with an acid. That is, a small amount of acid is added to such an extent that the dried peptide-solid conjugate is soaked, and the reaction is carried out at 20 to 80 ° C., preferably 30 to 60 ° C. for 5 minutes to 1 hour under a nitrogen atmosphere. As the acid, trifluoroacetic acid, heptafluorobutyric acid, hydrochloric acid-saturated acetic acid, or the like can be used, but trifluoroacetic acid, which has few side reactions, is most suitable. After the reaction is complete, add a peptide-soluble volatile solvent such as acetonitrile or propanol containing 0.1% trifluoroacetic acid, and then decompose and remove the solid phase to separate the desired carboxyl-terminal peptide into the liquid phase. To be taken. The thus-collected carboxyl-terminal peptide can have its structure determined by amino acid composition analysis and amino acid sequence analysis according to a conventional method, and can be used for other desired purposes.

〔The invention's effect〕

According to the present invention, the carboxyl-terminal peptide can be conveniently separated from the polypeptide, and the structure of the carboxyl-terminal of the polypeptide can be easily determined.

〔Example〕

 Examples of the present invention will be shown below.

Example 1 Egg white lysozyme was reacted with reduced trace monoiodoacetic acid with β-mercaptoethanol according to a conventional method to carboxymethylate the thiol group of the cysteine residue. 2 nmole of this carboxymethylated lysozyme was dissolved in 50 mMole of N-ethylmorpholine-acetate buffer (pH 9.0) containing 5 mole of urea, 10 picomole of API was added, and the mixture was mixed at 37 ° C for 6 minutes. Reacted for hours. After the reaction was completed, a 50 mM aqueous solution of N-ethylmorpholine was added to adjust the pH to 10.0.
Was adjusted to 50 mg, and 50 mg of DITC-CPG (controlled pore glass having phenylenediisothiocyanate bound thereto, manufactured by Sigma) was added, the reaction system was replaced with nitrogen, and the mixture was shaken at room temperature for 1 hour. Meanwhile, the coupling reaction was performed. After completion of the reaction, 50% acetonitrile / 2-propanol mixed solution containing 0.1% trifluoroacetic acid (volume ratio 3/7)
Washed in vacuo and dried in vacuum. After drying, 50 μ of trifluoroacetic acid was added and incubated at 40 ° C. for 15 minutes under a nitrogen atmosphere. A 50% acetonitrile / 2-propanol mixed solution (volume ratio 3/7) containing 0.1% trifluoroacetic acid was added to this, and the supernatant was collected by centrifugation and dried in vacuum. The carboxyl-terminal peptide thus obtained was analyzed by reverse phase high performance liquid chromatography. Using a 4.6mmφ × 250mm Bakerbond Octyl column, using acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as solvent, acetonitrile 0% to 60%
Elution was performed at a flow rate of 1 ml / min. The chromatogram obtained is as shown in Figure 1B),
Peak b was in agreement with the expected carboxyl-terminal peptide as a result of amino acid composition analysis (ninhydrin method) and amino acid sequence analysis (automatic Edman degradation method) (Table 1). (The amino-terminal residue of the peptide did not exist as expected.) Note that peak a and peak c were analyzed and no amino acid was detected, indicating that the peptide was not a peptide. In addition, FIG. 1A) is a chromatogram of the peptide mixture after API digestion.

Example 2 0.5 nmol of human serum albumin was digested with API in the same manner as in Example 1, and then the carboxyl-terminal peptide was fractionated in the same manner as in Example 1. The preparative peptide was analyzed in the same manner as in Example 1, and as a result, the peak C in FIG. 2B) was obtained.
Was analyzed and the expected amino acid composition value and amino acid sequence were shown. None of peaks a, b, d and e were peptides.

[Brief description of drawings]

FIG. 1 and FIG. 2 show reverse phase high performance liquid chromatography charts of the peptides treated in Example 1 and Example 2, respectively.

Claims (1)

[Claims] 1. A polypeptide is specifically cleaved at a peptide bond between a lysine residue in the polypeptide and a subsequent amino acid residue on the carboxyl terminal side, and the resulting peptide mixture is released on the surface. Is reacted with a solid having a functional group capable of reacting with an amino group of to form a covalent bond, and then the peptide bond between the amino terminal amino acid residue of each peptide and an adjacent amino acid residue is cleaved by acid treatment. Thus, a method for fractionating a carboxyl-terminal peptide is characterized in that the peptide that is released is collected.